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    Biomechanics

    (D-141) Fumarate Analog and Shc Inhibitor Do Not Improve Muscle Function or Fibrosis in Dystrophic Mice

    Friday, October 13, 2023
    9:30 AM – 10:30 AM PDT
    Location: Exhibit Hall - Row D - Poster # 141
    Introduction:: Sub-track: Biomechanics of Rehabilitation/Injury
    Duchenne muscular dystrophy (DMD) has increased extracellular matrix (ECM) components which can cause fibrosis and impaired muscle mechanical function. Previous studies have shown, that the quantity of ECM does not cause increased muscle stiffness, instead collagen cross-linking and alignment are more closely related to stiffness. We hypothesize that     a fumarate analog and Shc inhibitor, which have shown anti-fibrotic activity in other tissues, will reduce fibrosis within muscle and improve muscle stiffness in     mdx mice.  

    Materials and Methods:: 4-week-old wildtype (n=3) and dystrophic mdx mice (n=15) on DB2 background were used, since it has been shown to have more robust fibrotic phenotype. The wildtype mice and 5 mdx mice were given peanut butter without drugs. The remaining mdx mice were dosed with B110-3 (25mpk) (n=5), a Shc inhibitor, or fumarate analog IXC-109 (232mpk) (n=5) daily for 4 weeks before sacrifice. At sacrifice, soleus, tibialis anterior (TA), gastrocnemius (GA), diaphragm (DP), and heart were harvested. The left soleus underwent a passive mechanical stretch, an isometric twitch and tetanus, followed by 5 eccentric contractions to look at stiffness, specific tension, and damage caused by repeated eccentric contractions. The right sided limbs were pinned at approximate optimal length (Lo), covered in OCT, and frozen in isopentane cooled LN. The left sided limbs and heart were flash frozen in liquid nitrogen. The flash frozen soleus and DP were powdered, and underwent a hydroxyproline assay with collagen solubility to look at collagen content and collagen cross-linking. The soleus and DP in OCT were cut in 10um cross-sections using cryostat and were stained with Sirius red to investigate collagen fiber area and collagen packing density, or WGA and Hoescht to measure fiber size. All animal experiments were approved by the University of California, Davis Institutional Animal Care and Use Committee. 

    Results, Conclusions, and Discussions:: Results: Body weight was significantly lower in the mice treated with B110-3 compared to wildtype mice, but was not significantly different to the vehicle or IXC-109 treated mice. There was no difference in soleus muscle mass or length in either group. Muscle stiffness was not significantly different in the soleus between groups. Mdx mice tended to have lower tetanus stress, but were not significantly different than wildtype mice (Fig. 1A). Total collagen content was increased in mdx mice, significantly in both treatment groups, compared to wildtype mice in the diaphragm, but this was not seen in the soleus (Fig. 1B). Insoluble collagen, which is a measure of collagen cross-linking, was tended to be increased in the mdx diaphragms but was significantly higher in the mice treated with B110-3. There was no difference in crosslinking in the soleus. Sirius red staining revealed no change in collagen fiber area with treatment. There was also no significant difference in muscle fiber size between groups.  

    Conclusion: Treatment with B110-3 or IXC-109 did not significantly affect muscle passive or active mechanics. Collagen content was increased with in the mdx diaphragm, significantly in the treatment groups, compared to wildtype mice. IXC-109 treated mice also had higher insoluble collagen which indicates more collagen cross-linking.  Therefore, neither treatment appears to be an effective anti-fibrotic treatment for the DB2 mouse model of DMD. 


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